L-FORMS OF BACTERIA

TRANSLATION .O. 1018

a- February 1964

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L-FORM OF BACTERIA

[ Following is a translation of the Russian languagearticle by V. D. Timakov and G. Ya. Kagan, N. F. Gam-aleya Institute of Epidemiolcgy and Microbiology,AMN, USSR, which appeared in the Journal of the USSRAcademy of Medical Science, 15.11:25-38, 1960. Trans-lation performed by Sp/6 Charles T. Ostertag Jr.

L-forms of bacteria were first discovered in 1935 by Klieneberger whoisolated them from a culture of Streptobacillugs moniliformis and named themthe Ll-form in honor of the Lister Institute. In spite of the passage of 25years since the discovery of L-forms of bacteria, their biological nature,association with other forms of mutability, importance in the evolution ofmicroorganisms, and their role in infectious pathology are items of intensivestudy and dispute up to the present time.

In her first works Klieneberger clarified her proposed hypothesis,which proposed the L-form in a bacterial culture of Streptobacillus mon-iliformis was to be considered as one of the symbionts of two microorganisms(bacterial and peripneumonic), supponedly lacking in the mutual exchangeproducts for their own normal devel.pmento. In subsequent experimental workby Dienes et al., Klieneberger's hyrothesis was disproved when the authorssucceeded in proving that the L-forn is not a symbiont but a markedly changedvariant of a culture of Ztrmtnhtee$ 11-- m.

The bacterial origin of the L-form was corroborated in the works ofHeilman et al. and then even Klieneberger-Nobel, who having verified theresults of the experimental works of Dienes et al., renounced her ownhypothesis of symbiosis.

The biological nature of L-forms is discussed even in the present day.A. A. Imshenetskiy identifies the L-forms of bacteria with 'pettenkoferia"and other forms of mutation arising in response to unfavorable influences.[ The term "pettenkoferia" was used by Kuhn to designate the big "aberrentforms" which arose as the cells of a parasite of the bacteria - Kuhn, P. andSternberg, K., 1931 Ueber Bakterien und Pettenkoferien. Zentr. Bakt., I, Orig.121, 13-161. Cited from Bacterial Reviews i-15, 1950-51, p 93. ] In his

opinion the L-forms should be regarded as degenerate, cytopathological forms,destined for death. Other investigators (M. A. Peshkov; V. D. Timakov;G. Ya. Kagan; B. Ya. El'bert; Klieneberger-Nobel; Minsk; Tulasne and Brisou,and others) reject such an interpretation of the L-form, considering them asforms, highly stable in relation to the factors which caused their formationand emerging as a result of the adaptation of bacteria to changed conditionsof existence.

An analysis of the factual material presented in this article makes itpossible to reveal the biological nature of the L-forms of bacteria and differ-entiate them from other fo:= of mutation.

Variants, formed under the influence of various interactions, are relateto the L-forms of bacteria. Several amino acids are the most active L-trans-forming agents, for example, glycine, carboxylmethoxylamine, dl methionine,1 - phenylalanine and others; antibiotics - penicillin, for some species ofbacteria - streptomycin, bacteriophages, immune serum and complement.

Transformation of bacteria into L-forms depends on the species ofbacteria, status of the population, selection of transforming agent and theconditions of cultivation. Certain species of bacteria, for example,Streptobacillus moniliformnis, Pr. vulgar s, Salmonella, form L-forms more oftenthan others, for example, C. diphtheriae, or some pathogenic cocci (streptococciTogether with the factors causing the formation of L-forms in various speciesof bacteria (penicillin), factors are known, the L-transforming action ofwhich is strictly differential depending on the species; thus, for example,streptomycin, to which the L-forms of many species of bacteria are sensitive,is the L-transforming agent only for tubercular mycobacteria (Huertas).

The conditions of cultivation, contributing to the transformation ofbacteria into L-forms, are not the same for different species of bacteria.General conditions, regardless of species origin, are the need for normalhcrse sea..um or its substitutes (polyvinylpyrrolidone or vitamins of group B),which destroys the inhibitors to the growth of the L-form which are availablein common nutrient media, a semisolid or semiliquid consistency of the media,and an increase of their hypertonicity due to an augmentation of the osmoticregulators. Included as substences which stabilize the actions of t-nsformingagents are saccharose, MgS04, KH2PO4 and Na2 HP04, NaCl, KC1, CaCl2, LiC12 , andothers.

The presence of similar regulators in the medium is quite necessaryfor obtaining and cultivating L-forms of coccal species of bacteria; theirpresence is optional for Gram negative rod-shaped bacteria and is not requiredat all for such Gram positive rod-shaped bacteria as C. diphtheriae, which inthe presence of increased concentrations of NaCl, KCI, CaC12 and phosphate

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salts, rapidly disintegrates and perishes under the influence of the trans-forming agent, not having changed into L-forms (G. Ya. Kagan and V. T. Saven-kova).

A detailed analysis of experimental data received by us in the labo-ratory during the study of the L-forms of various pathogenic species of bac-teria (V. D. Timakov, G. Ya. Kagan, V. S. Levashev, S. G. Konm; V. D. Timakov,G. Yb. Kagan; G. Ya Kagan and V. S. Levashev; G. Ya Kagan and V. T. Savenikova;G. Ya. Kagan and Z. A. Pesina: V. S. Levashev; S. V. Prozorovskiy) and thematerials of other investit (A. A. ?e.i. -Ksi~ii; Klieneberger-Nobel; Dienes; Tulasne; Minsk; Taubeneck and itany others) show with certaintythat the L-forms of bacteria have a number of biological peculiarities,differentiating them from other forms of utabilitj.

L-forms of bacteria grow on semisolid (L.3N agar) and semiliquid(0.3% agar) nutrient media in the form of cha-acterIstic L-colonies with adelicate, transparent, broken periphery of a slimy consistency and a ratherdry, thickened, easily pigmented, center growing into the depth of the medium.Based on the form of the colonies, the L-forms of various species of bacteriaare the same. The dimensions of the colonies varj from 0.1-0.5 m in diameterup to 2-3 mm depending on the species of the bacteria and the cultivationmedium. In many species of bacteria, for example R P. mirabils.,E. Coli. V, Cholerae and Streptococcus hemolyticus, the L-colonies aredifferentiated into two types, these are the so-called "3B" and "3A" colonies(fig 1).

The "3" colonies have larger dimensz'.s thvii "3A" colonies. Duringprolonged storage they acquire a yellowish-brown col3r owing to the productionof the pigment - flavin. They adsorb the corresponding bacteriophages welland are lysed by them. They retain phage sensitive receptors and are agglut-inated by the serum of the original species of bacteria. They are subinoculatedin subcultures on media containing the tranformiIng agent and easily reverseinto the original culture of bacteria with -li-'-' r 'f the tr..nsfor. ligagent from the composition of the meditur. ' c.:-' .-> " -:-t; sz::ethirg llkean intermediate phase of L-transforation.

Type "3A" L-colonies form cultures of stabilizd L-f or-s and havesmaller dimensions than "3B" colonies. They lo nc - '. th2 correspondingbacteriophages since they lose the phage sensitive recert-nrz. Often they arenot agglutinated by the serum of the original species of baoteria. Vhey aresubinoculated in subcultures independent of the content o" -r=sforning agentin the medium. For the most part they do not revert into bacterial forms."3A" colonies represent the concluding phase of L-transformation. Accordingto microscopic structure "3A" and "3B" L-colonies are completely the same.

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Individual microstructural elements, components of the L-colony, representextremely peculiar analogs of cells, and, regardless of the sharply expressedpolymorphism, they are easily differentiated from any other forms of mutabil-ity. In the composition of L-colonies there are found spherical, bladder-like,sometimes pear shaped and branching forms from 1 up to 20 microns in sizeand of a different optical density, formatior of an uncertain, constantlychanging configuration which are dense and transparent and distinguished bya different refraction. There is a multitude of different vacuolozed formscontaining one or several vacuoles. In some species of bacteria - Pr. Vul-garis Ps. Fluorescens very long and thin flagellates are attached to thespherical bodies Kohler, Huertas). Submicroscopic filterable forms with asize from 100 up to 500 millimicrons are aLso constitutent microstructuralelements of L-colonies. They are located within the spherical, vacuolizedand other bodies in the form of inclusions, and also outside of these forma-tions in the form of loosely lying masses, forming the center of the L-colonygrowing into the agar. The submicroscopic, filterable forms have a sphericalellipsoidal form, are characterized by Brownian movement (Huertas), passthrough bacterial filters and are precipitated out during ultra centrifugation(lieneberger-Nobel, Tulasne, V. S. Levashev, 1960). All the microstructuralelements of L-forms are fixed poorly and are not stained by the Gram method.Together with the spherical bodies which are stained blue with the Giensastain and which contain red inclusions, there are bodies, always filled withnuclear substance, surrounded only by a narrow stria of basophilic cytoplazm(M. A. Peshkov, Nermut). As a result of the disruption of the cytoplasmaticand intensive nuclear division, the cytoplasm of large spherical and otherbodies is filled with submicroscopic granules of a nuclear origin (Tulasne,Nermut), as a result of which in the L-forms of bacteria there takes placea sharp reduction of the RNA/DNA coefficient, that is an increase in DNA.Separate L-elements of the "3A" type are distinguished from "30' by a changeof the chemical composition of the cell walls, by the loss of a-E diaminopi-melic acid (Taubeneck; Lederberg and St. Clair).

The morphogenesis of L-forms of bacteria is distinguished by the diversityof their development. In the initial phase of the tran3formation of bacterialcells into an L-form a disruption is noted in the function of division withthe preservation of growth, as a consequence of which bodies are formed whichare diverse in form and which grow to gigantic sizes. Thus in representativesof Gram negative rod-shaped bacteria most of all in the initial phase ofmorphogenesis, spherical bodies (fig 2) are formed; in some Gram positiverod-shaped bacteria, for example C. diphtheriae, long flagellates and bladderlike forms (fig 3); in hemolytic streptococci along with the formation ofspherical bodies there takes place, due to the destruction of the cell wall,the liberation of structureless granular masses of cellular contents, Mor-phogenesis of L-forms is concluded with the complete disappearance of bacterialcells of the initial species, which are replaced by the microstructural ele-ments of L-forms; L-colonies are formed which are no different in the external

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appearance and morphology of the individual microstructural elements in variousspecies of bacteria.(fig 14, 5, 6, 7).

A characteristic peculiarity of the L-forms of bacteria are the diversetypes of their multiplication. Individual microstructural elements of L-formsmultiply by simple division, division in various directions, budding (fig 8),and the formation of submicroscopic granular fo~ms which are capable of growinginto large spherical bodies which break up again into submicroscopic granv'lo.

The varied nature of multiplication and the reproducing capabilitiesof all microstructures of L-forms: Spheres, vacuolized bodies without aspecific' configuration and submicroscopic forms, determine the capabilityfor the prolonged preservation of the cultures in the L-form and ability topass into subcultures.

The prolonged subculturing of L-forms under conditions where a trans-formirg agent is present may be accompanied by 1) temporary stab'lization,that is the capability to be subinoculated in the L-form in media containingan L-transforming agent, 2) permanent stabilization, that is the cap~bilityto be subinoculated in an L-form independent of the content of trans"4rmingagent in the medium, and 3) by breaking up into submicroscopic filterable formspassivating in subcultures.

The stabilization of cultures in the L-form has been noted at thepresent time in manr species of bacteria: Pr jgal (Tulasne, V. S. Leva-shev), cholera, (Minsk, Lavillaurix), S (G. Ya. Kagan and V. S.Levashev), , htheri (G. Ya. Kagan and V. T. Savenkova), Staph. aureus(S. V. Prozorovskly), and Stretoc. hmovti (G. Ya. Kagan and V. S.Levashev).

A special feature of the chemistry of the L-form is the concentration ofsynthetic processes, directed to the preservation of the nuclear substance ofthe cell (Tulasne, M. A. Peshkov, Nermut); as we already pointed out a dis-ruption of cytoplasmic division occurs with the retention of nuclear division.This process involves a change in the composition of nucleic acid due to anincrease of the number of submicroscopic elements of the L-forms, consistingmainly of DNA, which are contained in all the microstructures of L-colonies(intracellular inclusions in large bodies and vacuolized forms, extracellularstructures forming the center of L-colonies, grain accumlations and struc-tureless masses, almost without exception consisting of submicroscopic gran-ules). In the process of L-trannformation, polysaccharide, protein and waterexchange varies. Together with the loss or inhibition of some fermentationsystems, inherent to the original species of bacteria, for example in theL-forms of Proteus vulgaris, S. t=rhosa or Staph. aureus (V. S. Levashev;V. D. Timakov, G. Ya. Kagan and V. S. Levashev; S. V. Prozorovskily), there iEobserved the acquisition of new systems, not characteristic to the original

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culture, for example the ability to ferment maltose (V. S. Levashev) andurine in the L-form of Proteus vulgaris (Minsk), to synthesize cholesteroland flavin or to promote anaerobic glycolysis (Minsk; Dienes). Changes tothe chemical structure and metabolism consequently lead to an increase inthe degree of stability to factors which cuased the formation of the L-form,for example to penicillin (V. S. Levashev, S. V. Prozorovskiy), carboxylmeth-oxylamine, glycine (Dienes), or streptomycin (Huertas) and also in changesof antigenic structure. The latter, maintaining species specificity andgeneral antigenic groupings with the initial cultures, changes. Thesechange- are expressed in the acquisition during the process of L-transformationof specific antigenic components (Tulasne, Weinberger, Madoff and Dienes;V. S. Levashev; V. D. Timakov, G. Ya. Kagan and V. S. Levashev; G. Ya Kaganand Ye. I. Koptelova, 1960).

A particular place in the problem of L-forms of bacteria belongs tothe investigation connected with the study of the L-forms' capability toreverse into bacterial cultures. It is difficult to overestimate the valueof this most important biological peculiarity of L-forms for biology andespecially for medicine. Reversion of L-forms into bacterial cultures takesplace sometimes without any kind of artificial influences on the population;such an origin of reversion may be conditionally called "spontaneous", it ismost often observed in freshly isolated cultures of L-forms which have notsucceeded in becoming stabilized (Dienes; M. A. Pesidov; G. Ya. Kagan andV. S. Levashev; S. V. Prozorovskiy). Artificially produced reversion isobserved most of all when the L-transforming agent is eliminated from thenutrient medium.

Morphological investigation of the process of reversion (V. D. Timakov,G. Ya. Kagan, V. S. Levashev, S. G. Komm; V. D. Timakov and G. Ya. Kagan-M. A. Peshkov; Tulasne; Kuhl and Liebermeister; Hopken and Bartman) testifiesto the multiple nature of the morphogenesis of reversion, which is approximatelythe same in L.-forms of various gram-negative species of bacteria. Reversiontakes place either by means of an elongation of large spherical bodies anuthe formation of beaker-shaped, fusiform, irregularly shaped or long windingcells, segmented into bacillary forms or by means of segmentation of largebodies and their disintegration into curved bacterial forms. The formationhas also been described of transitional branching forms from vacuoles whichbud off bacilli. Pictures 9-11 depict the film documentation of the processof reversion of L-forms of S. typhosa followed by us in a Fonbrune oil chamberby using time lapse motion picture filming. Reverse forms are mainly sphericalbodies which mcst often by the end of the first 24 hours are becoming elongated,converting into cells that are irregularly formed, or oblong or beaker-shaped;part of the cells die, the others become still more elongated, transformingin the course of 30- 0 hours into gigantic winding long forms (fig 10) whichare segmented and wh'ch bud off normal bacterial cells (fig 11).

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The reversion of L-forms of coccal, species has been studied littIr.A description of this process is encountered only in single works (S. V.Prozorovskiy; G. Ya. Kagan and V. S. Levashev). According to data of S. V.Prozorovskiy, the first phase of morphogenesis of reversion of L-forms ofStaphylococcus aureus is the gradual vacuolization of a large portion of thestructural elements of the L-colonies. Almost without exception, the latterconsist of large and medium size vacuoles, gradually filling ip with largegrains which are very close in external appearance to fine cocci. Thisprocess is concluded with the formation of coccal forms. The per;.ods ofvacuole regeneration of L-colonies and the formation of granular forms vary,they may be computed by days, weeks or months depending on the length of thereversion process in the individual strains of the L-form.

The above presented results of morphological investigations of thereversion process testify to its complexity and species variations. Allstructural elements of L-forms (winding forms, spherical and vacuolizedbodies, submicroscopic structures) possess a regenerative function capableof the reversion of bacterial cells. The regenerative function of all themicrostructures of L-forms and the multiple nature of reversion (segmentationof large bodies into bacillary forms, conversion into beaker shaped, fusiform,winding and other forms which bud off or break up into bacilli) in variousbacterial species testifies to the latitude of the adaptive functions ofbacteria, which in the L-form preservc the :apability for many methods ofreversion.

The reversion of L-forms depends on two conditions: On the conditionsof the media in which it is taking place and on the state of the populationof L-forms, that is the level of stabilization of the newly acquired featuresof L-cultures. The higher the level of stabilization, the more difficultand complicated the process of reversion into bacterial forms (data byG. Ya. Kagan and V. S. Levashev during the study of reversion of L-forms ofS. typhosa and Streptoc. haemolyticus, Minsk - Cholera vibrio; Tulasne -

Proteus vulgaris, S. V. Prozorovskiy - Staphylococcus aureus).

The difficulty of reversion of stabilized L-cultures is a classicexample of the stability of features acquired in the process of adaptation,since the longer the process of adaptation the more complicated and difficultthe return to the initial form. This is also supported by the nature ofregeneration of features of the initial cultures in the process of L-formreversion. Very noteworthy in this respect is the fact of the completeregeneration of all the biological features of the initial cultures includingfermentative, antigenic, pathogenic and sensitivity to the L-transformingagent in L-cultures of the first passages, for example in S. typhosa (G. YaKagan and V. S. Levashev), that is with less stabilization of "L-features".

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In studying the biological properties of bacterial cultures reversedfrom stabilized L-forms of the typhoid-fever causative agent, hemolyticstreptococcus and Staphylococcus aureus, it was established that completeregeneration of the features of the initial species didn't take place (G. YaKagan and V. S. Levashev; S.-V. Prozorovskiy).

The results:"are also interesting of other experiments carried out byin the laboratory (G. Ya. Kagan and V. S. Levashev, 1958). These testify tothe fact that in'cultures reversed from L-forms there is noted an expressedcapability toward L-transformation. They easily and rapidly produce formatiof L-colonies when inoculated on media containing penicillin. In this it musbe noted that alternate existence of cultures ii the L- and reversed bac-terial form leads to a greater and greater change of biological features ofthe latter:, to atypical fermentative and antigenic properties (fig 12).As is apparent from this.diagram, in contrast to the reverted cultures ob-ta-ined from the first transformed L-forms, completely reverting to the biological features of the initial type, cultures obtained upon reversion of L-formwhich have been transformed two and three times from reverted cultures, areatypical and do not regain the biological features 'of the initial speciesof bacteria..

In considering the nature of L-forms of bacteria, one cannot stop at,their connection with heteromorphic.variants, protoplasts, filterable forms,and also organisms similar to pleuropneumonia.

It was established by the comprehensive investigators, N. F. Gamaleya,M. P. Pokrovskiy, M. A. Peshkov, A4 A. Imshenetskiy, and others that underthe influence of unfavorable media factors (cultivation ac high and low temp-eratures on media containing incre4sed concentrations of carbohydrates orthe salts of Mg, Na, Li, and otherg) there sometimes are formed giant bacteriaforms growing in the shape of spheres, fusiform shapes and filaments whichN. F. Gamaleya designated as "heteromorphic". These forms are not connectedwith degenerative and involution forms, their formation in cultures is evidencby profound and specific changes in the nuclear apparatus of the bacteria (N.LGamaleya)'. M. A. Peshkov's point of view is close to N. G. Gamaleya"s.Peshkov's experimental works made it possible to classify heteromorphic growthinto forms of natural (Achromobactcr ep-t, ini) and forced polymorphism.

A. A. Imshenetskiy has a completely different interpretation of thenature of heteromorphic growth. While studying the nature of the influenceof concentrated solutions of several salts on individual representatives ofBacteriaceae and Bacilaceae, the author observed the appearance of giganticsphere like forms which were exposed to "vacuole degeneration" and disinte-gration into elements consisting of nuclear substances, The ultimate fateof these "reaction forms" is concluded either with destruction or a returnto the initial bacterial forms. The author considers heteromorphic growthas the effect of the degeneration of bacteria.*

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An analysis of experimental data obtained by us in the laboratorywhile studying heteromorphic variants of staphylococcus, gonococcus andC. diphtheriae (V. S. Prozorovskiy; 0. Ya. Kagan and A. A. Pes±na; G. Ya.Kagan and V. T. Savenkova) and the results of the works of M. A. Peshkovand other investigators do not allow that heteromorphic variants be con-sidered as cytopathological forms.

Heteromorphism from out point of v~ew is one of the forms of thebiological reaction3 of bacteria enabling their survival and preservationunder conditions of the temporary unfavorable influence of some physical,chemical or biological agents.

Heteromorphism is a temporary initial form of adaptation of bacteria,and is capable of converting into more perfect and constant forms of adapta-tion. A comparison of the biological features of heteromcrphic variantswith L-forms of bacteria testifies to the presence ef a similarity and dis-tinctions between them.

Heteromorphic variants compare with L-forms in a certain similarityof morphological structures and the formation of gigantic sphere-like formswhich are vacuolized and contain granules of a nucleated nature. In contrastto L-forms they originate in response to the short-termed action of smalldoses of destructively acting factors and are capable of prolonged cultiva-tion in subcultures; a cessation of the action leads to a rapid reversion ofbacterial forms of the initial species.

Observations of the formation process of L-forms by Staphylococcusaureus (S. V. Prozorovskiy) and gonococcus (G. Ya. Kagan and Z. A. Pesina)showed that, depending on the concentration of penicillin and salt stabilizersin the medium, there is observed a differentiating reaction of bacterialcultures, the formation of heteromorphic variants in low concentrations ofthe specified agents and the formation of L-forms at increased concentrationsand a lengthier effect. When heteromorphic variants of Staphylococcus aureusare passaged on media with increasing concentrations of penicillin it ispossible to obtain the growth of stabilized L-cultures (S. V. Prozorovskiy)

The prolonged passaging of heteromorphic variants of C. diphtheriaeon media with increasing concentrations of an L-transforming agent may beaccompanied by the conversion of part of the strains being passaged intoL-forms (G. Y. Kagan and V. T. Savenkova).

Thus the results of a comparative study of some biological featuresof heteromorphic and L-forms testifies to the fact that heteromorphic formsmay be seen as one of the initial transitional phases of development of L-forms of bacteria.

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In the-process of formation of L-forms regardless of the fact thatthey were formed as a result of the original reaction of the bacteria on cul-tivation under conditions of penicillin - serum - salt media or by meann ofthe gradual cultivation of heteromorphic forms on media with increased concen-trations of penicillin, the stability of the L-forms to penicillin increases10,000 times in comparison with the original culture. Along with this, rever-sion isn't observed on media with smaller concentrations of penicillin.

The resistance to penicillin of the originally obtained heteromorphicvariants of staphylococcus increases 10-50 times; their gradual passaging,without converting into L-forms, on media with increased concentrations ofantibiotics leads to an increase of resistance to penicillin, but never reachesthe degree of stability of L-fox=s. Contrary to the L-forms, as a measure ofincreasing the stability of heteromorphic variants, their cultivation on mediawith lower concentrations invariably is accompanied by reversion. With theexclusion of penicillin from the make-up of the medium, the reversion ofheteromorphic forms into coccal forms takes place in 4-5 days, L-formsconsiderably slower - from 1 to 5 months depending on the degree of stabili-zation of the culture in the L-form (S. V. Prozorovskiy).

Cultures which reverted from heteromorphic forms completely recoverthe properties of the initial cultures, including plasma coagulating, necrotoxicand virulent properties. Complete recover-I of the properties of initial staphy-lococcal cultures in the process of their reversion from L-forms were not ob-served. It should be noted here that the deg-ee of recovery of features is indirect connection with the level of stabilization of the culture in the L-form(S. V. Prozorovskiy).

Thus heteromorphic forms, which differ from L-forms by instabilityand lability of features newly acquired by them, easily reverse into the orig-inal species upon restoration of the initial conditions of cultivation andperish under the prolonged action of the factor which caused their formationin the cultures, or are converted under appropriated conditions into an L-formwhich is new according to its biological quality. Therefore heteromorphicforms should be considered as a temporary, fluctuating form of mutability ofmicroorganisms, which is not accompanied by stabilized changes which arepassed on through inheritance, but only temporarily an increased adaptabilityof the microorganism for a relatively short time to changing conditiors ofthe medium of inhabitation.

A comparison of the biological peculiarities of heteromorphic variantswith forms of an unfinished L- or M- cycle (Tulasne and Lavillaureix; Tulasneand krisou) testifies to their complete identity. They are similar accordingto conditions of formation, morphological properties, the ability for a rapidand complete reversion of bacterial forms upon the elimination of the actionof the transforming agent, and also they perish under a prolonging or strength-ening of its action.

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A prolonged passaging of these forms on media, containing the trans-forming factor may sometimes end with their conversion into L-forms.

The initial phase of the formation of L-forms of bacteria may be theformation of bacterial protoplasts. They, like L-forms of bacteria, areformed due to the actions which are blocking the synthesis of the dense cellwalls (Weibull; Park and Strominger). Thus the addition of lysozyme to aculture of Micrococcus lysodekticus or B. megatherium under conditions ofincreased osmotic pressure is accompanied by depolymerization and completedepletion of cell walls and the formation of spherical cells - lysozymeprotoplasts; during this, other vitally important cell functions are notaffected (Salton; Weibull).

The morphological similarity of penicillin protoplasts and L-formsis manifested in that the spherical dense bodies are not only a morphologicalexpression of protoplasts but also a constituent component of L-forms. Proto-plasts have the form of spherical bodies which increase in size during pro-longed incubation. Often '"moon-like" vacuoles are formed in them (Laderbergand St. Clair) and they take on the shape of a half-moon, made up of a densesubstance bordering on the side of a fully transparent vacuole. Within thevacuoles and dense spherical bodies, upon aging of the protoplasts, a greatnumber of dark granules are formed, for example in E. coli (Laderberg andSt. Clair), Ss_, and Strept. haemolvticus (G. Ya. Kaganand B. S. Levashev, 1960). The protoplasts, Just like the L-forms, do nottake the Gram stain and are distinguished by a certain osmotic friability,caused by the nature of the effect of penicillin on the cell wall. Possessingthe capability of assimilation, the protoplasts produce division forms thoughthey are not capable of subsequent regeneration, do not reproduce and arenot transferred into subcultures (Weibull; Klieneberger-Nobel).

Morphologically, L forms of bacteria are differentiated from protoplastsby a diversity of microstructures described above which are not detected inprotoplasts. The sphere-shaped bodies of L-forms, in contrast to protoplasts,reach very large dimensions and possess the ability to multiply and formcultures,, for example a culture of L-forms of V. cholerae (Lavillaureix),consisting mainly of sphere shaped bodies, and culturing for a long timein sub passages. The progressive passaging of protoplasts on solid nutrientmedia which contain an L-transforming agent may be completed with the forms-tion of L-cultures of the "3B" and "3A" types; L-forms have been obtainedfrom protoplasts of E. coli Pr. mirobilis, and S. Typhosa (Weibull, Leder-berg and St. Clair; Klieneberger-Nobel; Bohme and Taubenek; Landman, Altenbergand Ginoza; G. Ya. Kagan and V. Levashev, 1959). Thus, protoplasts, Justas the heteromorphic variants, appear as the initial phase of development ofthe L-forms of bacteria.

11

As we already pointed out, in the process of the formation of L-variants granular submicroscopic elements are formed. They are found insideof the larger bodies and outside of them. These elements pass through bac-terial filters. They form the center of L-colonies and are capable of repro-duction and reversion into bacterial forms. The problem of the connectionof these filterable elements of L-forms and filterable forms of bacteria isthe object of consideration in contemporary bacteriological literature (M. A.Peshkov; Huertas; KlLieneberger-Nobel).

A comparison of some biological features of filterable elements of theL-forms and filterable forms of bacteria testify to their biological nearness.Filterable forms of bacteria are morphologically analogous with filterableelements of L-forms. They have a spherical or oval form with a diameter from0.1 to 0.3 microns (B. N. Il'yashenko; V. D. Timakov, G. Ya. Kagan, Ye. I.Koptelova, N. N. Solov'yev; V. S. levashev, 1960, Juhasz, Lovas and Egyessy;Huertas). They pass through bacterial filters and precipitate out duringultra centrifugation (Klieneberger-Ilobel; Tulasne; Mandel and Terranova;V. S. Levashev, 1960). Filterable forms are made up mainly of DNA, but alsocontain RNA and other proteins (Juhasz; -V.-S. Levashev, 1960). Thus even inchemical composition they are also close to filterable elements of L-forms andare distinguished from them only by quantity. It is known that during thedisintegration of unchanged or mildly changed cells the number of filterableforms is not great; in heteromorphic variants they may be detected in the formof a considerable number of inclusions of a nucleate nature contained in agiant changed bacterial cell; they are observed in maximum numbers in L-formsof bacteria. A sharp increase of their number during the formation of L-formsof bacteria is conclusively shown in the experiments of Tulasne, Nermut andother authors.

Analysis and other biological features testify to the biologicalcommunity of filterable forms and filterable elements of L-forms of bacteria.Thus it is well known that when filtrates of L-forms are inoculated on theappropriate media it is possible to obtain the growth of L-colonies. V. S.Levashev (1960), when inoculating a filtrate of an aging culture of Pr. vulgarison a medium favorable for the growth of L-forms of the stated causative agent,was able to obta_.1 the growth of single colonies of L-forms.

Filterable elements of L-forms as well as filterable forms regeneratedinto bacterial cultures slowly and with difficulty. These cultures aredifferentiated by a retarded and sparse growth and by lowered fermentativeand antigenic properties and are considerabley different from the originalbacterial cultures. (V. D. Timakov, G. Ya. Kagan, Ye. I. Koptelova, N. N.Solov'yev; G. Ya. Kagan and V. S. Levashev).

12

In stmmarizing the comparative data, characterizing filterable ele-ments of L-forms and filterable forms of bacteria, their definite biologicalassociation must be noted. They are elementary reproducing and regeneratingunits of bacterial cells, the biological purpose of which is the preservationof the life of the bacteria under changed conditions of existence.

Morphologically and physiologically close to the L-forms of bacteriais the group of so-called pleuropneumonia-like organisms (PPLO). Thesemicroorganisms are different from bacteria, rickettsiae and viruses. They,Just like the L-forms of bacteria, grow on cell-less media which contain addi-tional substances: Normal serum, ascitic liquids, cholesterol, mucin,DNA, and others. PPLO colonies in external appearance, size and microscopicstructure are close to stabilized L-forms - colonies of the "3A" type. Thedimensions of the colonies vary from 250 up to 750 microns and higher. Theperiphery, as in L-forms, consists mainly of diverse, sphere-like and irregu-larly formed bodies and columnar structures; the center, which is growinginto the medium, consists of submicroscopic filterable granules and very finefilaments and is easily pigmented (Poetschke, Edward, Minck, Morton).

The various representatives of this group of microorganisms are morpho-logically similar and can hardly be distinguished from L-forms of bacteria.They are made up of microstructural elements of v=lous size and form, spherelike bodies, annular vacuolized and filamentous forms, and filterable granules.Myceliel structures are encountered more often in PPLO cultures than in L-forms (Freundt); filterable elements are distinguished by somewhat smallersizes - 100-150 millimicrons (Edward, Klieneberger-Nobel). The filtrationend point through Gradocol membrane filters for PPLO = 0.25 microns and forL-forms - 0.4 5 microns. The differences encountered in the details of micro-scopic structure and the sizes ol individual microstructural elements are soinsignificant that they cannot be considered as criteria for the differen-tiation -P individual species of PPLO from each cther and all of this groupfrom the L-forms of bacteria.

Just as L-forms of bacteria, PPLO are differentiated by Lhe multiplenature of reproduction, which takes place by means of segmentation of fila-mentous forms (Orskov, Dienes, Freundt), division and budding, and also bythe intracellular development of submicroscopic elements formed in the spherelike bodies and released during their disintegration (Klieneberger and Smiles).Electron microscope research (Klieneberger-Nobel, Norton) testifies to thefact that in many representatives of PPLO as well as in stabilized "3A" typeL-forms, the cell wall is lacking.

Based on the nature of growth and nutritive requirements, PPLO formsare close to L-forms of bacteria (Minck; Edward). Several species of PPLOs

13

are distinguished by the fact that they are lackin6 in supplementary nutritionfactors, for example yeast extract and especially cholesterol. Cholesterol,which is a vitally necessary growth factor of several PPLO forms, is notneeded for the growth of L-forms, which are capable of synthesizing it (MortonIn contrast to L-forms, PPLO forms grow well on appropriate nutrient mediaimmediately in the first inoculations. L-forms grow poorly in the first generations, going through a stage of adaptation to the nutrient medium. As thenumber of passages increases they grow more intensively.

The problem concerning the identity of PPLO forms and stabilized L-formof bacteria has been the object of dispute up to the present time (Edward,Klieneberger-Nobel, Minck, Morton). The community of the biological featuresof these groups of microorganisms makes it possible to speak about the commu-nity of their origin. In spite of the differences of some details of struc-ture and metabolism, they are basically identical not only according tomorphology, nature of growth and peculiarities of multiplication, but alsoby other features.

PPLO forms, like the stabilized L-forms, lose the fundamental componentof the cell wall - diaminopimelic acid (Kandler and Lehender). They are dis-tinguished by osmotic friability, sensitivity to streptomycin, aureomycinand terramycin, and a high stability to penicillin, thallium acetate and otherL-transforming agents (Dienes, Pulvertaft).

The basic distirction betwen PPLO forms and L-forms. of bacteria is theirlack of reversion into bacterial cultures. However even this difference hasonly a relative significance. Thus at the present time it is well known thatstabilized L-forms of many species of bacteria persistently lose the capabilityof reversion into the original species of bacteria. These stabilized L-formsare practically no different from PPLO forms. The feasibility has also beenestablished of the reversion of several species of PPLO forms into bacterialforms. For example in the experiments of Moustardier, Brisou and Perry, rever-sion was obtained into bacterial cultures of Streptoc. faecalis, B. faecalisalcaligenes and Pr. mirabilis of h strains of PPLO isolated from patients withnon-gonococcal urethritis. Wittler was also able to observe reversion of astrain of PPLO in a tissue culture into Corynebacterium.

Thus the community of the basic morphological and physiological featuresof PPLO forms and stabilized L-forms of bacteria testify to the fact that PPLOforms originate from L-forms, which appear as though they were a transitionalphase in the course of the evolutionary development of PPLO forms from bacteria.

Recognition of the biological community of PPLO forms and L-forms of

14

-An | | m n

bacteria is reflected in the classification system suggested by Tulasne andBrisou in accordance to which the basic criterium for the differentiation ofPPLO forms and L-forms is the fact of the proven bacterial origin of thelatter. In connection with this all the representatives of PPLO forms areregarded in the order Pleuropneumonialis, close to the family BorrelomycetaceaeParasitaceae and the genus Asterococcus (Borel). This order contains onlyone family Pleuropneumonieaceae and one genus Pleuropneumonia and is dividedinto the individual species: Pl. bovis, Pl. agalactiae. Pl. artitidis muris,Pl. cerebri muris, Pl. aviae, etc.

Acceptance of the biological community of PPLO forms and L-forms ofbacteria into the group Bacteropneumoniales has been abbreviated Bp. In thisgroup are treated the L-forms of all the bacterial species from which theywere obtained, for example: Bp. Proteus, Bp. Salmonella, Bp. Escherichia, etc.

The above presented results of the experimental investigations of thenature of L-forms of bacteria and their relation with other similar forms ofmutation permits the classification of these forms in relation to the systempresented below (fig 13).

According to this arrangement, all a'rms of mutation, emerging inresponse to the ii-fluence of various physical, chemical and biological agents,may be divided into the following three groups: 1. Variants with temporarilyaltered features (modified forms of mutation), 2. Variants with hereditarilyfixed altered features, 3. Degenerative-involution forms.

Heteromorphic or M-variants and subcellular units: Protoplasts, non-stabilized L-forms and submicroscopic filterable forms are regarded as variantswith temporarily altered features. Variants with temporarily altered featuresmay reverse into the initial species of bacteria and transform into stabilizedL-forms, these are temporary adjusted forms which ensure the survival of thebacteria in specific short-termed changes in the conditions of the medium.

Stabilized L-forms, pleuropneumonia like organisms or OTPP (PPLO orOTPP -- organisms of the peripneunonia type) and submicroscopic forms areregarded as variants with hereditarily fixed features.

Stabilized L forms originate from the above described variants withtemporarily changed features; in the course of their subsequent developmentand breeding they may produce the formation of new species of PPLO forms andalso disintegrate into submicroscopic subcultured forms which preserve theirvitality. Variants with heretically fixed altered features, including stab-ilized L-forms, are the most ideal form of adaptation of bacteria to changedconditions of existence, principally distinct from degenerative forms whichhave not developod adaptive arrangements and on the power of this are non viable.

15

PPLO is an example of the formation of new species during the processof the evolutionary development of bacteria. The basic driving forces con-trIbutlng to their origin in the course of evolution Is the formation of adaplye mechanisms and breeding which culminate in the formation of new speciesof PPLO.e

U 16

Figure captions (between pages 28 and 29)

1. "3A" and "30" type colonies of haemolytic streptococci.

2. Initial phase of the formation of L-forms.

3. Initial phase in the formation of L-forms in Str. haemolyticus. 1500x.

4. Microstructures of L-colonies of S. typhosa. 1500x.

5. Microstructures of L-colonies of 0. diphtheriae. 1500x.

6. Microstruct.*es of L-colonies of Streptococcus haemolyticus. 1500x.

7. Microstructures of L-colonies of Staphylococcus aureus. 1500x.

8. Multiplication of L-forms of khurpoz* by budding. See explanation inthe text. 1500x. [ *The word Xhurpoz is unknown. Presumably it ismeant to refer to a species of microorganisms, which can not be estab-lished. The explanation in the text doesn't clear anything up. ]

9. Goblet like cells of a form of reversion of L-forms of S. typhosa.1500x.

10. Cells of irregular forms and gigantic twisting forms of reversion ofL-forms of S. typhosa. 1500x.

Ui. Lengthy forms, segmenting and budding off normal bacterial cells ofthe L-form. 1500x.

17

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18

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19

34. Dienes, L., Proc. Soc. Exper. Biol. a Med., 1938, v 39, P 365.

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20

56. Idem, J. Gen Microbiol, 1949, V 3, P 434.

57. Idem, Ibid, 1951, v 5, p 525.

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61. Idem, Ibid, 1958, Bd, 173, S 376.

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69. Idem, Ibid, 1950, Bd. 156, S 181.

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71. Minck, R., Minck, A., Compt. rend. Soc. Biol., 1951, v 145, p 927.

72. Minck, R., Fruhling, L., Ibid., 1954, v 148, p 2091.

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75. Morton, H., In the Book: Bacterial and Mycotic Infections of Man,Philadelphia, 1959, P 557, 563.

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21

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86. Idem, Ibid, 1951, v 145, p 4e29.

87. Idem, Rev. in=uol., 1951, v 15, p 223.

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90. Idem,. Exper. Cell. Res., 1955, v 9, p 294.

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22

T "

In Typhus abdominalis N 1-1999

In Typhus abdominalis-N 6420

fli.YP. L . '%' L

In Typhus abdominalis F2

rnInitial cultureL~ Reversed cultures

with recovered properties' j Reversed cultures not

'- recovering properties

L-foms

Fig. 12. Reversion of features depending on alternate residence of thecultures in the L and bacterial form.

23.

Forms of mutation originating in re- Degenerativesponse to the infiuence of various phys- involutionicalt chemical and biological factors.] forms

Variant with temporarily Variants with hereditarillyaltered features (modified fixed altered feature~sforms of mutation)

Hetero-I Proto- rNon Submicr,morphic plasts stable, scopic

or ML-formnR filt era'

_________ StabiliZedl Origination_______ L-forms i fnew

D B

RevesionintoSubmicroscopicinitil spciessubinoculatedbacteriaforms

Figure 13. Biological reactions of bacteria in response to the' influenceof physical, chemical, and biological factors.

24e